Method for preparing and purifying polypeptides

A purification method and polypeptide technology, applied in the field of medicine, can solve the problems such as no peptide amino acid oxide, no targeted research on methionine oxidation impurities, and no effective detection.

Active Publication Date: 2020-02-28
LUNAN PHARMA GROUP CORPORATION
View PDF8 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this impurity cannot be effectively detected by the general peptide detection method, so the methionine oxidation impurity has not been studied in a targeted manner
There is no detailed research on polypeptide oxides in the current polypeptide drug preparation literature and patents, and there is no case where polypeptide amino acid oxides are listed as impurities in the quality standards

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for preparing and purifying polypeptides
  • Method for preparing and purifying polypeptides
  • Method for preparing and purifying polypeptides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] The preparation and purification of embodiment 1 ularitide

[0082] (1) Dissolution and oxidation of the crude product of ularitide linear peptide

[0083]Take 10.0 g of the crude linear peptide, stir and dissolve it with 10.0 L, pH 8.0, 1 mol / L ammonium acetate aqueous solution, and oxidize it with stirring air at 25°C. After 6 hours of oxidation, the content of cysteine ​​residues is detected to be 0, and the oxidation is stopped. ; HPLC detects the oxidation solution, and the HPLC purity of ularitide is 51.81%, and the methionine oxidation impurity (relative retention time=0.61) is 2.24% ( figure 1 ).

[0084] (2) Concentration of oxidizing solution

[0085] Concentrate 10.0 L of the ularitide oxidation solution obtained in step (1) by nanofiltration, stop the concentration when the volume reaches 1.0-1.2 L, and adjust the pH of the concentrated solution to 6.0 with acetic acid for later use.

[0086] (3) Purification by high performance liquid chromatography

[...

Embodiment 2

[0094] The preparation and purification of embodiment 2 ularitide

[0095] (1) Dissolution and oxidation of the crude product of ularitide linear peptide

[0096] Take 10.0 g of crude linear peptide, stir and dissolve it with 10.0 L, pH 6.0, 1.5 mol / L aqueous solution of potassium dihydrogen phosphate, and oxidize it with stirring air at 20°C. After 8 hours of oxidation, the content of cysteine ​​residues is detected to be 0. , stop oxidation; HPLC detection of oxidation solution, HPLC purity of ularitide is 52.12%, methionine oxidation impurity is 2.15%.

[0097] (2) Concentration of oxidizing solution

[0098] 10.0 L of the ularitide oxidation solution obtained in step (1) was applied to a D152 cation exchange resin column, desalted and eluted with 0.1 N hydrochloric acid aqueous solution, and the elution volume was 1.0-1.2 L for later use.

[0099] (3) Purification by high performance liquid chromatography

[0100] DAC50 column is used, the water circulation outside the ...

Embodiment 3

[0107] The preparation and purification of embodiment 3 ularitide

[0108] (1) Dissolution and oxidation of the crude product of ularitide linear peptide

[0109] Take 10.0 g of the crude linear peptide, stir and dissolve it with 10.0 L, pH 6.5, 5.0 mol / L ammonium chloride aqueous solution, and oxidize it in air with stirring at 30 ° C. After 5 h of oxidation, the content of cysteine ​​residues is detected to be 0. Oxidation was stopped; the oxidized solution was detected by HPLC, and the HPLC purity of ularitide was 51.78%, and the methionine oxidation impurity was 2.18%.

[0110] (2) Concentration of oxidizing solution

[0111] Concentrate 10.0 L of the ularitide oxidation solution obtained in step (1) by nanofiltration, stop the concentration when the volume reaches 1.0-1.2 L, and adjust the pH of the elution concentrate to 6.0 with dilute hydrochloric acid for later use.

[0112] (3) Purification by high performance liquid chromatography

[0113] DAC50 column is used, t...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention belongs to the technical field of medicine, and particularly discloses a method for preparing and purifying polypeptides. A method for inhibiting methionine oxidation is used in the preparation and purification process of ularitide, so that the purity of the purified refined ularitide is as high as 99.90%, and methionine oxidation impurity peptides are controlled within 0.1%.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a method for preparing and purifying polypeptides. Background technique [0002] The structure of Uralitide is a polypeptide composed of 32 amino acids containing a pair of intramolecular disulfide bonds, which is generally prepared by chemical synthesis. The sequence structure is: [0003] H-Thr-Ala-Pro--Arg-Arg-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser -Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-OH (S-S). [0004] Ularitide is a natriuretic peptide isolated from human urine. It was first isolated from urine by SchulzKnappe et al. in 1988 and belongs to the atrial natriuretic peptide (ANP) family. Renal natriuretic peptide. Endogenous uraritide is synthesized in the distal renal tubular cells, secreted after the lumen, and binds to the downstream natriuretic peptide A receptor in the inner medullary collecting duct, which can regulate t...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/58C07K14/575C07K1/20
CPCC07K14/58C07K14/57572
Inventor 张贵民刘东李铁健
Owner LUNAN PHARMA GROUP CORPORATION
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap